You Youwen, Proctor Rachel M, Vasilko Eric D, Robinson Mary A
School of Veterinary Medicine, Department of Clinical Studies, University of Pennsylvania, New Bolton Center Campus, Kennett Square, Pennsylvania, 19348, USA.
PA Equine Toxicology & Research Laboratory, 220 East Rosedale Avenue, West Chester, Pennsylvania, 19382, USA.
Drug Test Anal. 2019 May;11(5):649-658. doi: 10.1002/dta.2542. Epub 2018 Dec 9.
JWH-250 is a synthetic cannabinoid. Its use is prohibited in equine sport according to the Association of Racing Commissioners International (ARCI) and the Fédération Équestre Internationale (FEI). A doping control method to confirm the presence of four JWH-250 metabolites (JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, JWH-250 5-OH-indole, and JWH-250 N-pentanoic acid) in equine urine was developed and validated. Urine samples were treated with acetonitrile and evaporated to concentrate the analytes prior to the analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The chromatographic separation was carried out using a Phenomenex Lux 3 μm AMP column (150 x 3.0 mm). A triple quadrupole mass spectrometer was used for detection of the analytes in positive mode electrospray ionization using multiple reaction monitoring (MRM). The limits of detection, quantification, and confirmation for these metabolites were 25, 50, and 50 pg/mL, respectively. The linear dynamic range of quantification was 50-10000 pg/mL. Enzymatic hydrolysis indicated that JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, and JWH-250 5-OH indole are highly conjugated whereas JWH-250 N-pentanoic acid is not conjugated. Relative retention time and product ion intensity ratios were employed as the criteria to confirm the presence of these metabolites in equine urine. The method was successfully applied to post-race urine samples collected from horses suspected of being exposed to JWH-250. All four JWH-250 metabolites were confirmed in these samples, demonstrating the method applicability for equine doping control analysis.
JWH - 250是一种合成大麻素。根据国际赛马委员会协会（ARCI）和国际马术联合会（FEI）的规定，其在马术运动中被禁止使用。已开发并验证了一种用于确认马尿液中四种JWH - 250代谢物（JWH - 250 4 - 羟基戊基、JWH - 250 5 - 羟基戊基、JWH - 250 5 - 羟基吲哚和JWH - 250 N - 戊酸）存在的兴奋剂检测方法。尿液样本用乙腈处理并蒸发以浓缩分析物，然后通过液相色谱 - 串联质谱法（LC - MS/MS）进行分析。色谱分离使用Phenomenex Lux 3μm AMP柱（150×3.0 mm）进行。使用三重四极杆质谱仪在正模式电喷雾电离下通过多反应监测（MRM）检测分析物。这些代谢物的检测限、定量限和确证限分别为25、50和50 pg/mL。定量的线性动态范围为50 - 10000 pg/mL。酶水解表明JWH - 250 4 - 羟基戊基、JWH - 250 5 - 羟基戊基和JWH - 250 5 - 羟基吲哚高度共轭，而JWH - 250 N - 戊酸未共轭。相对保留时间和产物离子强度比被用作确证马尿液中这些代谢物存在的标准。该方法已成功应用于从疑似接触JWH - 250的马匹收集的赛后尿液样本。在这些样本中确认了所有四种JWH - 250代谢物，证明了该方法在马兴奋剂检测分析中的适用性。
